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1.  Regulation of Gene Expression of Catecholamine Biosynthetic Enzymes in Dopamine-β-Hydroxylase- and CRH-Knockout Mice Exposed to Stress 
Norepinephrine-deficient mice harbor a disruption of the gene for dopamine-β-hydroxylase (DBH-KO). Corticotropin-releasing hormone knockout mice (CRH-KO) have markedly reduced HPA activity. The aim of the present work was to study how deficiency of DBH and CRH would affect tyrosine hydroxylase (TH), DBH, and phenylethanolamine N-methyltransferase (PNMT) gene expression and protein levels in the adrenal medulla (AM) and stellate ganglia (SG) of control and stressed mice. Both in AM and SG, single immobilization significantly increased TH and DBH mRNA and protein levels both in wild-type (WT) and CRH-KO mice. On the other hand, the stress-triggered increase in the PNMT mRNA and protein levels seen in WT mice was absent in CRH KO mice. DBH-KO mice are more sensitive to stress but survive a single 2 h restraint stress in a tube (RES). The increase in TH mRNA levels induced by RES in WT was not observed in DBH-KO mice. PNMT mRNA and especially PNMT protein levels were significantly elevated in AM of DBH-KO mice. In SG of DBH-KO mice TH mRNA levels were not affected; however, PNMT gene expression was highly elevated. Thus, disruption of the DBH gene surprisingly blocks the stress-induced elevation of TH mRNA levels in AM but increases PNMT gene expression both in AM and SG. Our data indicate that adrenergic signaling is required for stress-induced increase in TH mRNA and that this signaling restrains stress-induced increase in PNMT mRNA. They also confirm that the HPA system plays a crucial role in the stress-induced regulation of PNMT gene expression.
PMCID: PMC4482230  PMID: 19120118
catecholamine biosynthetic enzymes; gene expression; DBH- and CRH- knockout mice; restraint stress; immobilization; glucocorticoid regulation
2.  Passion for Participatory Research on the Menstrual Cycle 
A two-day symposium entitled “The Menstrual Cycle and Adolescent Health” was held in Potomac, Maryland in mid October 2007. Groups sponsoring the meeting included the Department of Health and Human Services (DHHS), the National Institutes of Health (NIH), the National Institute of Child Health and Human Development, the American Society for Reproductive Medicine, the NIH Office of Research on Women’s Health, the NIH Office of Rare Diseases, the US Food and Drug Administration Office of Women’s Health, the DHHS Office of Women’s Health, and Rachel’s Well, Inc. Attendees included patients, patient advocates, and experts from a variety of fields and disciplines. The effort identified areas in which there are only sparse data from which to create evidence-based recommendations for management of menstrual issues in young adolescents. In a final session of the meeting, which is the subject of this report, participants worked together to develop a manifesto regarding research on the menstrual cycle in adolescents. The group reached two major conclusions. First, there is need for a new research model that integrates grass roots community passion for participatory research in the research planning and regulatory oversight. Secondly, there is a need for a coordinated research effort on the menstrual cycle and its disorders in adolescents. This could initially take the form of a Study of Puberty Across the Nation (SPAN), similar to the Study of Women Across the Nation (SWAN) that addressed the normal menopausal process.
PMCID: PMC4455018  PMID: 18574237
adolescents; menstrual cycle; research models
3.  Mitochondrial and Nuclear Cross Talk in Cell Death: Parthanatos 
Poly(ADP-ribose) polymerase-1 (PARP-1) PARP-1 is an abundant nuclear protein first described to facilitate DNA base excision repair. Recent work has expanded the physiologic functions of PARP-1 and it is clear that the full range of biologic actions of this important protein are not yet fully understood. Regulation of the product of PARP-1, poly(ADP-ribose) (PAR), is a dynamic process with poly(ADP-ribose) glycohydrolase (PARG) playing a major role in the degradation of the polymer. Under pathophysiologic situations, over activation of poly(ADP-ribose) polymerase-1 (PARP-1) results in unregulated PAR synthesis and widespread neuronal cell death. Once thought to be necrotic cell death due to energy failure, we recently found that PARP-1 dependent cell death is dependent on the generation of PAR that triggers nuclear translocation of apoptosis-inducing factor (AIF) to result in caspase-independent cell death. This form of cell death is distinct from apoptosis, necrosis or autophagy and is termed Parthanatos. PARP-1 dependent cell death has been implicated in tissues throughout the body and in diseases afflicting hundreds of millions world wide including stroke, Parkinson's disease, heart attack, diabetes, and ischemia reperfusion injury in numerous tissues. The breadth of indications for PARP-1 injury make Parthanatos a clinically important form of cell death to understand and control.
PMCID: PMC4454457  PMID: 19076445
4.  Neuropeptides, Mesenteric Fat, and Intestinal Inflammation 
The ability of fat tissue cells to produce proinflammatory cytokines and the concept that obesity represents a low-grade inflammatory response have been well documented during the past decade. The effects of fat-mediated inflammation on metabolic pathologies have also been drawing increasing interest, However, very little is known on the potential effects of adipose tissue in the pathophysiology of gastrointestinal diseases with an inflammatory component, such as Inflammatory Bowel Disease (IBD). The development of large fat masses around the inflamed intestine during Crohn’s disease makes this tissue a candidate for more intense investigation in studies aiming to gain insights in the pathogenesis and progress of the disease. Furthermore, neuropeptides act in many cases in a proinflammatory manner and are shown to participate in the pathogenesis of intestinal inflammation in animal models of IBD However, the potential of these molecules to interact with fat cells in the context of IBD has not been investigated. In this review we describe our most recent data related to the effects of neuropeptides on non-inflammatory fat tissue components. In addition, we include discussion to associate neuropeptide-induced, adipose tissue-mediated responses with the generation of intestinal inflammatory conditions such as Crohn’s disease.
PMCID: PMC4404029  PMID: 19076372
Neuropeptides; Substance P (SP); Neurotensin (NT); Intestinal inflammation; Inflammatory Bowel Disease (IBD); Adipose tissue; Mesenteric fat; “creeping fat”
5.  Associations between sleep loss and increased risk of obesity and diabetes 
During the past few decades, sleep curtailment has become a very common behavior in industrialized countries. This trend for shorter sleep duration has developed over the same time period as the dramatic increase in the prevalence of obesity and diabetes. There is rapidly accumulating evidence to indicate that chronic partial sleep loss may increase the risk of obesity and diabetes. Laboratory studies in healthy volunteers have shown that experimental sleep restriction is associated with an adverse impact on glucose homeostasis. Insulin sensitivity decreases rapidly and markedly without adequate compensation in beta cell function, resulting in an elevated risk of diabetes. Prospective epidemiologic studies in both children and adults are consistent with a causative role of short sleep in the increased risk of diabetes. Sleep curtailment is also associated with a dysregulation of the neuroendocrine control of appetite, with a reduction of the satiety factor leptin and an increase in the hunger-promoting hormone ghrelin. Thus, sleep loss may alter the ability of leptin and ghrelin to accurately signal caloric need, acting in concert to produce an internal misperception of insufficient energy availability. The adverse impact of sleep deprivation on appetite regulation is likely to be driven by increased activity in neuronal populations expressing the excitatory peptides orexins that promote both waling and feeding. Consistent with the laboratory evidence, multiple epidemiologic studies have shown an association between short sleep and higher body mass index after controlling for a variety of possible confounders.
PMCID: PMC4394987  PMID: 18591489
Sleep Deprivation; Diabetes; Obesity; Glucose Tolerance; Energy Expenditure; Epidemiology; leptin; ghrelin; appetite; orexins
6.  Behavioral Effects of Cb2 Cannabinoid Receptor Activation And Its Influence On Food and Alcohol Consumption 
Consumers of marijuana typically feel a strong compulsive desire to consume food. Although past research revealed that the CB1 cannabinoid receptor is a potent regulator of food intake, the functional presence of neuronal CB2 cannabinoid receptors in the brain had been controversial. The role of CB2 receptors in food and alcohol consumption and the behavioral effects of CB2 receptor ligands are not well characterized. This is because CB2 cannabinoid receptors were thought to be absent from the brain and expressed primarily in immune cells and in the periphery. We tested the effects of peripheral injections of CB2 antagonist AM 630, CB2 agonist PEA and CB1 antagonist AM 251 on male C57BL/6, Balb/c, and DBA/2 mice at the beginning of the night cycle and after overnight 12-hour fasts. We also investigated the effects of the putative CB2 agonist, JWH015, and CB2 antagonist SR144528 in mouse motor function tests and in the two compartment black and white box. Under standard conditions, the CB2 antagonist AM 630 inhibited food consumption in C57BL/6 mice and DBA/2 mice, but failed to block food intake of Balb/c mice. The CB2 agonist PEA had no significant effect on food consumption in Balb/c mice, and reduced food intake in C57BL/6 and DBA mice. The CB1 antagonist AM 251 inhibited food ingestion in the three mouse strains at variable times. After 12-hour food deprivation, the CB2 antagonist AM 630 increased food consumption in C57Bl/6 mice, but failed to produce significant changes in food intake for Balb/c and DBA/2 mice. The CB2 agonist PEA also reduced food consumption in all three mice strains at variable times. In comparison to the CB2 ligands, CB1 antagonist AM 251 inhibited food ingestion in the mouse strains. A general pattern of depression in locomotor activity was induced by JWH 015 in both males and females in the three mouse strains tested as the dose was increased. The development and enhancement of alcohol preference was observed following chronic treatment with CB2 agonist JWH 015 in stressed mice but not in controls. Using the DBA/2 strain the spontaneous locomotor activity and stereotype behavior was enhanced by acute administration of low doses of SR144528. There was a reduction in CNR2 gene expression in the ventral mid-brain region of mice that developed alcohol preference but not in those that did not develop alcohol preference. These effects of CB2 cannabinoid receptor ligands in in vivo behavioral tests are provided as functional evidence that CB2-Rs in the brain plays a role in food and alcohol consumption and in the modification of mouse behavior.
PMCID: PMC4219871  PMID: 18991890
Food intake; alcohol consumption; cannabinoids; CB1 and CB2 cannabinoid receptors; CB2-R gene expression; locomotor activity; stereotypy
7.  Molecular genetics of addiction and related heritable phenotypes: genome wide association approaches identify “connectivity constellation” and drug target genes with pleiotropic effects 
Genome wide association (GWA) can elucidate molecular genetic bases for human individual differences in “complex” phenotypes that include vulnerability to addiction. Here, we review: a) evidence that supports polygenic models with (at least) modest heterogeneity for the genetic architectures of addiction and several related phenotypes; b) technical and ethical aspects of importance for understanding genome wide association data: genotyping in individual samples vs DNA pools, analytic approaches, power estimation and ethical issues in genotyping individuals with illegal behaviors; c) the samples and the data that shape our current understanding of the molecular genetics of individual differences in vulnerability to substance dependence and related phenotypes; d) overlaps between GWA datasets for dependence on different substances; e) overlaps between GWA data for addictions vs other heritable, brain-based phenotypes that include: i) bipolar disorder, ii) cognitive ability, iii) frontal lobe brain volume, iv) ability to successfully quit smoking, v) neuroticism and vi) Alzheimer’s disease. These convergent results identify potential targets for drugs that might modify addictions and play roles in these other phenotypes. They add to evidence that individual differences in the quality and quantity of brain connections make pleiotropic contributions to individual differences in vulnerability to addictions and to related brain disorders and phenotypes. A “connectivity constellation” of brain phenotypes and disorders appears to receive substantial pathogenic contributions from individual differences in a constellation of genes whose variants provide individual differences in the specification of brain connectivities during development and in adulthood. Heritable brain differences that underlie addiction vulnerability thus lie squarely in the midst of the repertoire of heritable brain differences that underlie vulnerability to other common brain disorders and phenotypes.
PMCID: PMC3922196  PMID: 18991966
pleiotropic; cell adhesion; monte carlo
Major depression and addiction are mental health problems associated with stressful events in life with high relapse and reoccurrence even after treatment. Many laboratories were not able to detect the presence of CB2 cannabinoid receptors (CB2-Rs) in healthy brains, but there has been demonstration of CB2-R expression in rat microglial cells and other brain associated cells during inflammation. Therefore, neuronal expression of CB2-Rs had been ambiguous and controversial and its role in depression and substance abuse is unknown. In this study we tested the hypothesis that genetic variants of CB2 gene might be associated with depression in a human population and that alteration in CB2 gene expression may be involved in the effects of abused substances including opiates, cocaine and ethanol in rodents. Here we demonstrate that a high incidence of (Q63R) but not (H316Y) polymorphism in the CB2 gene was found in Japanese depressed subjects. CB2-Rs and their gene transcripts are expressed in the brains of naïve mice and are modulated following exposure to stressors and administration of abused drugs. Mice that developed alcohol preference had reduced CB2 gene expression and chronic treatment with JWH015 a putative CB2-R agonist, enhanced alcohol consumption in stressed but not in control mice. The direct intracerebroventricular microinjection of CB2 anti-sense oligonucleotide into the mouse brain reduced mouse aversions in the plus-maze test, indicating the functional presence of CB2-Rs in the brain that modifies behavior. Using electron microscopy we report the sub cellular localization of CB2-Rs that are mainly on post-synaptic elements in rodent brain. Our data demonstrate the functional expression of CB2-Rs in brain that may provide novel targets for the effects of cannabinoids in depression and substance abuse disorders beyond neuro-immunocannabinoid activity.
PMCID: PMC3922202  PMID: 18991891
Neuronal CB2 Cannabinoid Receptors; Brain; electron micrograph; chronic mild stress; anhedonia; depression; drug abuse
9.  Basic and Clinical Immunology of Siglecs 
Siglecs are cell-surface proteins found primarily on hematopoietic cells. By definition, they are members of the immunoglobulin gene super-family and bind sialic acid. Most contain cytoplasmic tyrosine motifs implicated in cell signaling. This review will first summarize characteristics common and unique to Siglecs, followed by a discussion of each human Siglec in numerical order, mentioning in turn its closest murine ortholog or paralog. Each section will describe its pattern of cellular expression, latest known immune functions, ligands, and signaling pathways, with the focus being predominantly on CD33-related Siglecs. Potential clinical and therapeutic implications of each Siglec will also be covered.
PMCID: PMC3902170  PMID: 19076345
Siglec; human; mouse; leukocyte; ITIM; ITAM; sialic acid; lectin
10.  Retinoic Acid in the Immune System 
Annals of the New York Academy of Sciences  2008;1143:10.1196/annals.1443.017.
On occasion, emerging scientific fields intersect and great discoveries result. In the last decade, the discovery of regulatory T cells (Treg) in immunity has revolutionized our understanding of how the immune system is controlled. Intersecting the rapidly emerging field of Treg function, has been the discovery that retinoic acid (RA) controls both the homing and differentiation of Treg. Instantly, the wealth and breadth of knowledge of the molecular basis for RA action, its receptors, and how it controls cellular differentiation can and will be exploited to understand its profound effects on Treg. Historically, vitamin A deprivation and repletion and RA agonists have been shown to profoundly affect immunity. Now these findings can be interpreted in light of the revelations that RA controls leukocyte homing and Treg function.
PMCID: PMC3826166  PMID: 19076350
vitamin A; retinoic acid; immune system; tolerance
11.  The Expression of NMDA Receptor Subunit mRNA in Human Chronic Alcoholics 
Annals of the New York Academy of Sciences  2008;1139:10.1196/annals.1432.053.
Ethanol is a modulator at the N-methyl-d-aspartate class of glutamate receptors in the brain. In animal studies the receptor adapts to sustained ethanol exposure through altered expression of the subunits that make up the receptor complex. We used real-time RT-PCR normalized to GAPDH to assay NR1, NR2A, and NR2B subunit mRNA in superior frontal and primary motor cortex tissue obtained at autopsy from chronic alcoholics with and without co-morbid cirrhosis of the liver, and from matched controls. The expression of all three subunits was significantly lower in both areas of cirrhotic alcoholics than in the corresponding areas in both controls and alcoholics without co-morbid disease, who did not differ significantly from each other. The decrease was area-dependent when cases were partitioned by the 5-HTTLPR allele. Thus, polymorphisms in one gene can have a significant effect on the expression of a second, unrelated, gene. The expression of the N-methyl-d-aspartate glutamate receptor complex is under multifactorial control.
PMCID: PMC3821866  PMID: 18991843
5-HTTLPR; autopsy; glutamate; excitotoxicity; cerebral cortex; genotype–phenotype interactions
12.  Studying the Brain Gut Axis with Pharmacological Imaging 
Annals of the New York Academy of Sciences  2008;1144:10.1196/annals.1418.025.
Pharmacological imaging provides great potential both for evaluating the efficacy of new candidate compounds in the treatment of gastrointestinal symptom-based disorders, and for furthering our understanding of the underlying pathophysiology of such disorders. By combining evaluation of symptoms, behavior, and brain responses to relevant stimuli, use of neuroimaging is able to move the study of brain-gut disorders away from more subjective outcomes and emphasize the underlying neural networks involved in symptom generation and treatment. This chapter reviews the state of the art in pharmacological imaging studies, both in human subjects and in animal models of brain gut interactions.
PMCID: PMC3817712  PMID: 19076383
Irritable Bowel Syndrome; Brain-Gut axis; neuroimaging; functional gastrointestinal disorder
13.  Role of Increased ROS Dissipation in Prevention of T1D 
Annals of the New York Academy of Sciences  2008;1150:10.1196/annals.1447.045.
Protection of pancreatic β cells is an approach to prevent autoimmune type 1 diabetes (T1D) and to protect transplanted islets. Reactive oxygen species (ROS) are important mediators of β cell death during the development of T1D. We have examined the role of elevated ROS dissipation in the prevention of T1D using the ALR mouse strain. The selection of ALR, for resistance against alloxan-induced free radical–mediated diabetes, led to a strain of mice with an elevated systemic as well as pancreatic ROS dissipation. Independent genetic mapping studies have identified ALR-derived diabetes protective loci. Conplastic and congenic mouse as well as cell line studies have confirmed the genetic mapping and demonstrated that the elevated ROS dissipation protects ALR β cells from autoimmune destruction. Our data support the hypothesis that elevated ROS dissipation protects β cells against autoimmune destruction and prevents T1D development.
PMCID: PMC3817822  PMID: 19120287
type 1 diabetes; reactive oxygen species; mitochondria; mouse model; genetics
14.  Menstrual Cycle Dysfunction Associated with Neurologic and Psychiatric Disorders 
Epilepsy, bipolar disorder, and migraines are common disorders that are often associated with disturbances in menstrual function in adolescent girls. Women with untreated epilepsy are more likely to have irregular menstrual cycles than are nonepileptic controls, indicating that the disease itself plays a role in the etiology of these reproductive abnormalities. In addition, many girls with these disorders require chronic maintenance treatment with agents that may perturb the hypothalamic-pituitary-ovarian axis. Valproate is a highly effective antiepileptic drug used widely to treat epilepsy, bipolar disorder, and migraines. Valproate induces features of the polycystic ovary syndrome (PCOS) in approximately 7% of women. Girls with epilepsy, and possibly bipolar disorder, appear particularly susceptible to developing PCOS features on valproate, perhaps on account of the relative immaturity of their hypothalamic-pituitary-ovarian axes. Antipsychotics are highly effective drugs used widely to treat adolescents with bipolar disorder, psychotic disorders, and behavioral disturbances. Some, but not all of the antipsychotic, induce hyperprolactinemia, which may result in oligo- or amenorrhea. Prolonged amenorrhea in association with hyperprolactinemia incurs significant risks for bone health in adolescent girls. Because of the potential reproductive health risks associated with use of specific antiepileptic drugs and selective antipsychotics, these agents are vital treatments for adolescents with severe illnesses. Use of these agents should be considered and weighed against the risk of using alternative agents, which have their own side effects, or not treating these serious neurologic and psychiatric disorders.
PMCID: PMC3773702  PMID: 18574228
bipolar disorder; epilepsy; hypothalamic-pituitary-gonadal (HPG) axis; menstrual cycle dysfunction; migraines; polycystic ovarian syndrome (PCOS); valproate
15.  Cerebral White Matter 
Lesions of the cerebral white matter (WM) result in focal neurobehavioral syndromes, neuropsychiatric phenomena, and dementia. The cerebral WM contains fiber pathways that convey axons linking cerebral cortical areas with each other and with subcortical structures, facilitating the distributed neural circuits that subserve sensorimotor function, intellect, and emotion. Recent neuroanatomical investigations reveal that these neural circuits are topographically linked by five groupings of fiber tracts emanating from every neocortical area: (1) cortico-cortical association fibers; (2) corticostriatal fibers; (3) commissural fibers; and cortico-subcortical pathways to (4) thalamus and (5) pontocerebellar system, brain stem, and/or spinal cord. Lesions of association fibers prevent communication between cortical areas engaged in different domains of behavior. Lesions of subcortical structures or projection/striatal fibers disrupt the contribution of subcortical nodes to behavior. Disconnection syndromes thus result from lesions of the cerebral cortex, subcortical structures, and WM tracts that link the nodes that make up the distributed circuits. The nature and the severity of the clinical manifestations of WM lesions are determined, in large part, by the location of the pathology: discrete neurological and neuropsychiatric symptoms result from focal WM lesions, whereas cognitive impairment across multiple domains—WM dementia—occurs in the setting of diffuse WM disease. We present a detailed review of the conditions affecting WM that produce these neurobehavioral syndromes, and consider the pathophysiology, clinical effects, and broad significance of the effects of aging and vascular compromise on cerebral WM, in an attempt to help further the understanding, diagnosis, and treatment of these disorders.
PMCID: PMC3753195  PMID: 18990132
fiber tracts; neuropsychiatry; cognition; demyelination; vascular dementia
16.  The Placental Syncytium and the Pathophysiology of Preeclampsia and Intrauterine Growth Restriction 
Preeclampsia is associated with an increased release of factors from the placental syncytium into maternal blood, including the antiangiogenic factors soluble fms-like tyrosine kinase-1 and soluable endoglin, the antifibrinolytic factor plasminogen activator inhibitor-1, prostanoids, lipoperoxides, cytokines, and microparticles. These factors are suggested to promote maternal endothelium dysfunction and are associated with placental damage in pregnancies also complicated with intrauterine growth restriction (IUGR). In this report, we briefly describe the interaction of syncytial factors with hypoxia, reactive oxygen species, and apoptosis in the pathophysiology of preeclampsia and IUGR. Given the critical role of the syncytium in these complications of pregnancy, we also present a novel methodology in which laser capture microdissection followed by Western blotting is used to assess levels of syncytial Fas ligand, a key protein in the apoptotic cascade.
PMCID: PMC3671376  PMID: 18443340
Placenta; syncytiotrophoblast; reactive oxygen species; apoptosis; Fas ligand; pathophysiology of preeclampsia; IUGR; laser capture microdissection
17.  Corticosterone Regulates pERK1/2 Map Kinase in a Chronic Depression Model 
Neurotransmitter- or neurotrophin-regulated intracellular signaling in the hippocampus is hypothesized to contribute to depression and antidepressant (ADT) efficacy. Extracellular signal-regulated kinase 1/2 (ERK1/2) is downstream of several receptor types and regulates transcriptional activity of many targets; ERK1/2 may thereby influence mood and affect. Using a novel, ADT-sensitive depression model in mice, we show that prior corticosterone exposure decreases motivated behavior, sucrose consumption, and pERK1/2 in the dentate gyrus, but not in CA1/CA3. Notably, prefrontal cortical targets were also regulated. Our data suggest ADTs restore hippocampal pERK1/2 after stress-related insult, and potentially reveal a novel role for prefrontal neurotrophins in depressive-like symptomology.
PMCID: PMC3657205  PMID: 19120149
stress; antidepressant; anhedonia; prefrontal cortex; BDNF; dentate gyrus; amitriptyline; fluoxetine
18.  Monitoring the State of Cholecystokinin Receptor Oligomerization after Ligand Binding Using Decay of Time-Resolved Fluorescence Anisotropy 
Oligomeric complexes of G protein–coupled receptors (GPCRs) are now commonly recognized and can provide a mechanism for regulation of signaling systems. Receptor oligomerization has been most extensively studied using coimmunoprecipitation and bioluminescence or fluorescence resonance energy-transfer techniques. Here, we have utilized decay of time-resolved fluorescence anisotropy of yellow fluorescent protein-labeled cholecystokinin receptor constructs to examine the state of oligomerization of this receptor in living cells. The rotational correlation times established that the cholecystokinin receptor is constitutively present in an oligomeric state that is dissociated in response to agonist occupation. In contrast, antagonist occupation failed to modify this signal, leaving the oligomeric structure intact. This dynamic technique complements the other biochemical and steady-state fluorescence techniques to establish the presence of oligomeric receptor complexes in living cells.
PMCID: PMC3580951  PMID: 19076359
G protein–coupled receptors; cholecystokinin receptor; receptor oligomerization; time-resolved anisotropy; rotational dynamics
19.  Vitamin C–mediated Maillard Reaction in the Lens Probed in a Transgenic-mouse Model 
Aging human lens crystallins are progressively modified by yellow glycation, oxidation, and cross-linked carbonyl compounds that have deleterious properties on protein structure and stability. In order to test the hypothesis that some of these compounds originate from oxidized vitamin C, we have overexpressed the human vitamin C transporter 2 (hSCVT2) in the mouse lens. We find that levels of ascorbic and dehydroascorbic acid are highly elevated compared to the wild type and that the lenses have accumulated yellow color and advanced Maillard reaction products identical with those of the human lens. Treatment of the mice with nucleophilic inhibitors can slow down the process, opening new avenues for the pharmacological prevention of senile cataractogenesis.
PMCID: PMC3485640  PMID: 18448816
glycation; ascorbic acid; crystallin; cross-linking; aging
20.  Recent Structural and Mechanistic Insights into Endplate Acetylcholine Receptors 
Voluntary movement mediated by skeletal muscle relies on endplate acetylcholine receptors (AChR) to detect nerve-released ACh and depolarize themuscle fiber. Recent structural and mechanistic studies of the endplate AChR have catalyzed a leap in our understanding of the molecular steps in this chemical-to-electrical transduction process. Studies of acetylcholine binding protein (AChBP) give insight into ACh recognition, the first step in activation of the AChR. An atomic structural model of the Torpedo AChR at a resolution of 0.4 nm, together with single-ion channel recording methods, allow tracing of the link between the agonist binding event and gating of the ion channel, as well as determination of how the channel moves when it opens to allow flow of cations. Structural models of the human AChR enable precise mapping of disease-causing mutations, while studies of the speed with which single AChR channels open and close cast light on pathogenic mechanisms.
PMCID: PMC3478106  PMID: 18567853
acetylcholine receptor; acetylcholine binding protein; agonist recognition; binding-gating coupling mechanism; congenital myasthenic syndrome
21.  Further Observations in Congenital Myasthenic Syndromes 
During the past five years many patients suffering from congenital myasthenic syndromes (CMS) have been identified worldwide and novel causative genes and mutations have been discovered. The disease genes now include those encoding each subunit of the acetylcholine receptor (AChR), the ColQ part of acetylcholinesterase (AChE), choline acetyltransferase, Nav 1.4, MuSK, and Dok-7. Moreover, emerging genotype-phenotype correlations are providing clues for targeted mutation analysis. This review focuses on the recent observations in selected CMS.
PMCID: PMC3478107  PMID: 18567859
congenital myasthenic syndromes; acetylcholinesterase; choline acetyltransferase; acetylcholine receptor; Dok-7
22.  Lymphatic Involvement in Lymphangioleiomyomatosis 
Lymphangioleiomyomatosis (LAM) is a rare, multisystem disease affecting primarily premenopausal women. The disease is characterized by cystic lung disease, at times leading to respiratory compromise, abdominal tumors (in particular, renal angiomyolipomas), and involvement of the axial lymphatics (e.g., adenopathy, lymphangioleiomyomas). Disease results from the proliferation of neoplastic cells (LAM cells), which, in many cases, have a smooth muscle cell phenotype, express melanoma antigens, and have mutations in one of the tuberous sclerosis complex genes (TSC1 or TSC2). In the lung, LAM cells found in the vicinity of cysts are, at times, localized in nodules and may be responsible for cyst formation through the production of proteases. Lymphatic channels, expressing characteristic lymphatic endothelial cell markers, are found within the LAM lung nodules. LAM cells may also be localized within the walls of the axial lymphatics, and, in some cases, penetrate the wall and proliferate in the surrounding adipose tissue. Consistent with extensive lymphatic involvement in LAM, the serum concentration of VEGF-D, a lymphangiogenic factor, is higher in LAM patients than in healthy volunteers.
PMCID: PMC3392168  PMID: 18519973
lymphangiogenesis; metastasis; lymphangioleiomyomatosis; VEGF-D; VEGF-C
23.  Rebooting the Immune System with High-Dose Cyclophosphamide for Treatment of Refractory Myasthenia Gravis 
A small but important proportion of patients with myasthenia gravis (MG) are refractory to conventional immunotherapy. We have treated 12 such patients by “rebooting” the immune system with high-dose cyclophosphamide (Hi Cy, 200 mg/kg), which largely eliminates the mature immune system, while leaving hematopoietic precursors intact. The objective of this report is to describe the clinical and immunologic results of Hi Cy treatment of refractory MG. We have followed 12 patients clinically for 1–9 years, and have analyzed their humoral and cellular immunologic parameters. Hi Cy is safe and effective. All but one of the patients experienced dramatic clinical improvement for variable periods from 5 months to 7.5 years, lasting for more than 1 year in seven of the patients. Two patients are still in treatment-free remission at 5.5 and 7.5 years, and five have achieved responsiveness to immunosuppressive agents that were previously ineffective. Hi Cy typically reduced, but did not completely eliminate, antibodies to the autoantigen AChR or to tetanus or diphtheria toxin; re-immunization with tetanus or diphtheria toxoid increased the antibody levels. Despite prior thymectomy, T cell receptor excision circles, generally considered to reflect thymic emigrant T cells, were produced by all patients. Hi Cy treatment results in effective, but often not permanent, remission in most refractory myasthenic patients, suggesting that the immune system is in fact “rebooted,” but not “reformatted.” We therefore recommend that treatment of refractory MG with Hi Cy be followed with maintenance immunotherapy.
PMCID: PMC3390145  PMID: 18567882
myasthenia gravis; refractory MG; high-dose cyclophosphamide; Hi Cy; rebooting the immune system; TRECs; autoimmunity; immunotherapy
24.  Endometriosis and Tissue Factor 
Tissue factor (TF), is a cellular receptor that binds the ligand factor VII/VIIa to initiate the blood coagulation cascade. In addition to its role as the initiator of the hemostatic cascade, TF is known to be involved in angiogenesis via an interaction with factor VIIa and protease-activated receptor-2 (PAR-2). In this article we review previous studies from our laboratory demonstrating that the pattern and level of TF expression is altered in multiple cell types derived from eutopic and ectopic endometrium from women with endometriosis compared with normal endometrium. We posit that the inflammatory environment that occurs in ectopic and eutopic endometrium from patients with disease results in high TF expression that in turn, signals via PAR-2 to further produce inflammatory cytokine or chemokine production and macrophage recruitment. Thus, our studies suggest that TF might be an ideal target for therapeutic intervention in endometriosis.
PMCID: PMC3129033  PMID: 18443336
endometriosis; endometrium; tissue factor
25.  Stem Cells and the Pathogenesis of Endometriosis 
Endometriosis is a common gynecological disorder that is defined by the presence of endometrial tissue outside the uterine cavity. This disease often results in extensive morbidity, including chronic pelvic pain and infertility. The pathogenesis of endometriosis is likely multifactorial, and extensive investigation has explored the role of genetics, environmental factors, and the immune system in predisposing patients to developing endometriosis. A series of recent publications have described the identification of endometrial stem/progenitor cells. Such cells have long been speculated to function in the cyclic regeneration of the endometrium during the menstrual cycle and in the pathogenesis of several gynecological disorders. This narrative review will (i) examine the evidence for endometrial stem cells, (ii) examine their potential role in the pathogenesis of endometriosis, and (iii) identify important unanswered questions with suggestions for future investigation.
PMCID: PMC3107843  PMID: 18443337
Endometrium; endometriosis; stem cells; bone marrow; uterus

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